Cytochromes P450 (CYPs) play an active role in the metabolism of numerous physiological substrates such as steroid hormones, fatty acids, prostaglandins and bile acids, as well as countless xenobiotics, including drugs, chemical carcinogens, insecticides, petroleum products, and other environmental pollutants. Oxidative metabolism catalyzed by CYP can result in detoxification. However, in some instances it results in metabolic activation of a chemical to cytotoxic and/or carcinogenic form. Certain forms of CYP are associated with disease. For example, CYP2E1 is associated with liver disease since treatment with diallyl sulfate which suppresses CYP2E1 prevents liver disease.
Characterization of cytochrome P450, a superfamily of more than 160 known members (Nelson et al., 1993), continues to provide valuable information about protein structure-function relationships and the regulation of gene expressions. The human and rodent genomes contain at least 50 P450 genes which are classified into at least ten families according to amino acid sequence data (Gotoh, 1992).
A nomenclature system for the P450 system has been set forth in Nelson et al. (1993) and will be used herein unless otherwise noted. Briefly, for the gene and cDNA, the italicized root symbol "CYP" for humans and all nonhuman species except mice, representing "cytochrome P450" is used (in mice the root is Cyp). This root is followed by an Arabic number denoting the family, a letter designating the subfamily and an Arabic numeral representing the individual gene within the subfamily. The same nomenclature is also used for the mRNA and protein product of the gene without italicization. Alternative nomenclature is also used either (1) drop the root designation and use only the alphanumeric designation following the root CYP or (2) replace the root with P450.
The term form-specific refers to antibody that is specific for one form, i.e. an enzyme that is coded for by a specific gene within a subfamily. The form-specific antibody appears to be directed to a unique epitope on the enzyme, one that is not found on other forms of CYP. The term inhibitory refers is used to indicate antibody that inhibits the catalytic activity of the enzyme.
The enzymes encoded for by these genes show partial sequence overlap but distinct substrate specificities. Much work has been directed to determining which parts of a P450 protein are involved in recognition and binding of substrates and hence determining substrate specificity (Gotoh, 1992).
CYP2B1 has 97% identity with the primary sequence of CYP2B2 (Guengerich et al., 1982; Waxman and Walsh, 1982; Waxman et al., 1983), but they are products from distinct genes. Only 16 amino acids out of approximately 500 residues are different. However, these 16 amino acid substitutions render 2B1 different from 2B2 with respect to substrate specificity and catalytic rate. CYP2B1 exhibits an approximately 5-fold higher catalytic activity toward benzphetamine (Guengerich et al., 1982) and testosterone (Waxman and Walsh, 1982) than does CYP2B2. Further, 2B1 has an approximately seven-fold greater catalytic activity than 2B2 toward pentoxyresorufin (Lubet et al., 1985). However, 2B2 has a much higher catalytic activity than 2B1 in formation of methylhydroxylidocaine from lidocaine (Oda et al., 1989).
One of the main tools in studies of cytochrome P450 are polyclonal and monoclonal antibodies directed against the CYPs. The similarity in their primary sequences, however, leads to cross-reactivity of polyclonal antibodies to other forms in the same subfamilies even following extensive affinity purification. Furthermore, polyclonal or monoclonal antibodies produced against whole CYP molecules are generally not inhibitory because binding of the antibodies to antigenic sites on CYPs do not necessarily inhibit the catalytic activities of the CYPs.
Antipeptide antibody production against synthetic peptides was determined to be an efficient method for the production of specific antibodies against CYPs (Frey et al., 1985; Edwards et al., 1990; Edwards et al., 1991a and 1991b; Friedberg et al., 1991; Murry et al., 1993). The versatility in the selection of epitopes for antipeptide antibody production facilitated generation of antibodies directed against epitopes involved in catalytic activity of CYPs. In general, antipeptide antibodies so far produced against CYPs are not form-specific nor completely inhibitory toward catalytic activities (Frey et al., 1985; Edwards et al., 1990; Edwards et al., 1991a and 1991b; Friedberg et al., 1991; Murry et al., 1993).
Friedberg et al. (1991) reported that antipeptide antibodies, produced against a recombinant fusion peptide of CYP2B1 or CYP2B2 and a P450 unrelated protein, were neither form-specific nor inhibitory. Usually, antipeptide antibodies produced against recombinant fusion peptides are not inhibitory because these antibodies do not recognize the native peptides (Friedberg, 1991).
It would be useful to have antibodies which are form-specific to identify CYPs which are involved in the metabolism of non-physiological compounds/substances, such as drugs and chemicals or physiological substances. Further, it would be useful to be able to inhibit a specific CYP either to allow the use of a drug which would normally be degraded by the CYP or conversely to prevent the metabolic activation of a chemical to a toxic form.